Background: In-home iron fortification for infants in developing countries is recommended for control of anaemia, but low absorption typically results in >80% of the iron passing into the colon. Iron is essential for growth and virulence of many pathogenic enterobacteria. We determined the effect of high and low dose in-home iron fortification on the infant gut microbiome and intestinal inflammation. Methods: We performed two double-blind randomised controlled trials in 6-month-old Kenyan infants (n=115) consuming home-fortified maize porridge daily for 4 months. In the first, infants received a micronutrient powder (MNP) containing 2.5 mg iron as NaFeEDTA or the MNP without iron. In the second, they received a different MNP containing 12.5 mg iron as ferrous fumarate or the MNP without the iron. The primary outcome was gut microbiome composition analysed by 16S pyrosequencing and targeted real-time PCR (qPCR). Secondary outcomes included faecal calprotectin (marker of intestinal inflammation) and incidence of diarrhoea. We analysed the trials separately and combined. Results: At baseline, 63% of the total microbial 16S rRNA could be assigned to Bifidobacteriaceae but there were high prevalences of pathogens, including Salmonella Clostridium difficile, Clostridium perfringens, and pathogenic Escherichia coli. Using pyrosequencing, +FeMNPs increased enterobacteria, particularly Escherichia/Shigella (p=0.048), the enterobacteria/bifidobacteria ratio (p=0.020), and Clostridium (p=0.030). Most of these effects were confirmed using qPCR; for example, +FeMNPs increased pathogenic E. coli strains (p=0.029). +FeMNPs also increased faecal calprotectin (p=0.002). During the trial, 27.3% of infants in +12.5 mgFeMNP required treatment for diarrhoea versus 8.3% in −12.5 mgFeMNP (p=0.092). There were no study-related serious adverse events in either group. Conclusions: In this setting, provision of iron-containing MNPs to weaning infants adversely affects the gut microbiome, increasing pathogen abundance and causing intestinal inflammation. Trial registration number: NCT01111864.

Iron supplements acutely increase hepcidin, but the duration and magnitude of the increase, its dose dependence and effects on subsequent iron absorption have not been characterized in humans. Better understanding of these phenomena might improve oral iron dosing schedules. We investigated whether the acute iron-induced increase in hepcidin influences iron absorption of successive daily iron doses and twice daily iron doses. We recruited 54 non-anemic young women with plasma ferritin ≤20 µg/l and conducted: 1) a dose-finding investigation with 40, 60, 80, 160 and 240 mg labeled Fe as [⁵⁷Fe]-, [⁵⁸Fe]- or [⁵⁴Fe]-FeSO₄ given at 8.00 fasting on one or on two consecutive days (study 1, n=25; study 2, n=16); and 2) a study giving three 60 mg Fe doses (twice a day dosing) within 24h (study 3, n=13). In studies 1 and 2, 24hr after doses of 60 mg or greater, serum hepcidin was increased (P<0.01) and fractional iron absorption was decreased by 35-45% (P<0.01). With increasing dose, fractional absorption decreased (P<0.001), while absolute absorption increased (P<0.001). A six-fold increase in iron dose (40 to 240 mg), resulted in only a 3-fold increase in iron absorbed (6.7 to 18.1 mg). In study 3, total iron absorbed from three doses (two mornings and afternoon) was not significantly greater than that from two morning doses. Providing lower dosages (40-80 mg Fe) and avoiding twice a day dosing maximizes fractional absorption. The duration of the hepcidin response supports alternate day supplementation, but longer-term effects of these schedules require further investigation. These clinical trials were registered at www.clinicaltrials.gov as NCT01785407 and NCT02050932.

In iron-depleted women without anemia, oral iron supplements induce an increase in serum hepcidin (SHep) that persists for 24 hours, decreasing iron absorption from supplements given later on the same or next day. Consequently, iron absorption from supplements is highest if iron is given on alternate days. Whether this dosing schedule is also beneficial in women with iron-deficiency anemia (IDA) given high-dose iron supplements is uncertain. The primary objective of this study was to assess whether, in women with IDA, alternate-day administration of 100 and 200 mg iron increases iron absorption compared to consecutive-day iron administration. Secondary objectives were to correlate iron absorption with SHep and iron status parameters. We performed a cross-over iron absorption study in women with IDA (n=19; median hemoglobin 11.5 mg/dL; mean serum ferritin 10 mg/L) who received either 100 or 200 mg iron as ferrous sulfate given at 8 AM on days 2, 3 and 5 labeled with stable iron isotopes 57Fe, 58Fe and 54Fe; after a 16-day incorporation period, the other labeled dose was given at 8 AM on days 23, 24 and 26 (days 2, 3 and 5 of the second period). Iron absorption on days 2 and 3 (consecutive) and day 5 (alternate) was assessed by measuring erythrocyte isotope incorporation. For both doses, SHep was higher on day 3 than on day 2 (P<0.001) or day 5 (P<0.01) with no significant difference between days 2 and 5. Similarly, for both doses, fractional iron absorption (FIA) on days 2 and 5 was 40-50% higher than on day 3 (P<0.001), while absorption on day 2 did not differ significantly from day 5. There was no significant difference in the incidence of gastrointestinal side effects comparing the two iron doses (P=0.105). Alternate day dosing of oral iron supplements in anemic women may be preferable because it sharply increases FIA. If needed, to provide the same total amount of iron with alternate day dosing, twice the daily target dose should be given on alternate days, as total iron absorption from a single dose of 200 mg given on alternate days was approximately twice that from 100 mg given on consecutive days (P<0.001). In IDA, even if hepatic hepcidin expression is strongly suppressed by iron deficiency and erythropoietic drive, the intake of oral iron supplements leads to an acute hepcidin increase for 24 hours. The study was funded by ETH Zürich, Switzerland. This study has been registered at www.clinicaltrials.gov as #NCT03623997.

Iron deficiency and iron deficiency anemia (IDA) are major public health problems worldwide, especially in young women. Oral iron supplementation can be an effective strategy to treat and prevent IDA, but guidelines vary. Some experts recommend doses of 150-200 mg elemental iron per day, with the dose split through the day. However, recent studies suggest this may not be an optimal regimen. The fraction of iron absorbed from high doses of oral iron is low, and unabsorbed iron can cause gut irritation, inflammation and dysbiosis, and these reduce compliance. In recent studies using serum hepcidin profiles and stable iron isotopes to quantify iron absorption in young women, we have shown that: (a) oral iron doses ≥60 mg in iron-deficient women, and doses ≥100 mg in women with IDA, stimulate an acute increase in hepcidin that persists 24 h after the dose, but subsides by 48 h; (b) therefore, to maximize fractional iron absorption, oral doses ≥60 mg should be given on alternate days; (c) the circadian increase in plasma hepcidin is augmented by a morning iron dose; therefore, iron doses should not be given in the afternoon or evening after a morning dose. If rate of Hb response is important, a pooled analysis of our data done for this review indicates that total iron absorption is also higher if twice the target daily iron dose is given on alternate days. In summary, these studies suggest changing from daily to alternate-day schedules and from divided to morning single doses increases iron absorption and may reduce side effects. Thus, providing morning doses of 60-120 mg iron as a ferrous salt given with ascorbic acid on alternate days may be an optimal oral dosing regimen for women with iron-deficiency and mild IDA.

Objective: Iron-containing micronutrient powders (MNPs) reduce anaemia in African infants, but the current high iron dose (12.5 mg/day) may decrease gut Bifidobacteriaceae and Lactobacillaceae , and increase enteropathogens, diarrhoea and respiratory tract infections (RTIs). We evaluated the efficacy and safety of a new MNP formula with prebiotic galacto-oligosaccharides (GOS) combined with a low dose (5 mg/day) of highly bioavailable iron. Design: In a 4-month, controlled, double-blind trial, we randomised Kenyan infants aged 6.5–9.5 months (n=155) to receive daily (1) a MNP without iron (control); (2) the identical MNP but with 5 mg iron (2.5 mg as sodium iron ethylenediaminetetraacetate and 2.5 mg as ferrous fumarate) (Fe group); or (3) the identical MNP as the Fe group but with 7.5 g GOS (FeGOS group). Results: Anaemia decreased by ≈50% in the Fe and FeGOS groups (p<0.001). Compared with the control or FeGOS group, in the Fe group there were (1) lower abundances of Bifidobacterium and Lactobacillus and higher abundances of Clostridiales (p<0.01); (2) higher abundances of virulence and toxin genes (VTGs) of pathogens (p<0.01); (3) higher plasma intestinal fatty acid-binding protein (a biomarker of enterocyte damage) (p<0.05); and (4) a higher incidence of treated RTIs (p<0.05). In contrast, there were no significant differences in these variables comparing the control and FeGOS groups, with the exception that the abundance of VTGs of all pathogens was significantly lower in the FeGOS group compared with the control and Fe groups (p<0.01). Conclusion: A MNP containing a low dose of highly bioavailable iron reduces anaemia, and the addition of GOS mitigates most of the adverse effects of iron on the gut microbiome and morbidity in African infants. Trial registration number: NCT02118402.